U.S. patent application number 11/634908 was filed with the patent office on 2008-06-12 for nanoparticles composed of alkyl-cyanoacrylate polymers.
This patent application is currently assigned to TONG SHEN ENTERPRISE CO., LTD.. Invention is credited to Chi-Yu Huang, Yu-Der Lee.
Application Number | 20080138418 11/634908 |
Document ID | / |
Family ID | 39498351 |
Filed Date | 2008-06-12 |
United States Patent
Application |
20080138418 |
Kind Code |
A1 |
Lee; Yu-Der ; et
al. |
June 12, 2008 |
Nanoparticles composed of alkyl-cyanoacrylate polymers
Abstract
The role of nanoparticle composition as biodegradable carriers
for variously therapeutical drugs is disclosed. Nanoparticles are
synthesized by anion emulsion polymerization of two
alkyl-cyanoacrylate monomers with adjusted content ratio. By
modulating the compositions, particle size, hydrophobicity and
degradation rate of the copolymers is controlled. Hence, to
encapsulate wide range of therapeutical drugs, poly(alkyl
cyanoacrylate) nanoparticles with feasible compositions are applied
individually. The copolymer nanoparticles produced by n-butyl
cyanoactylate (BCA) and 2-octyl cyanoacrylate (OCA), for example,
were used therein. The nanoparticles composed of poly[(n-butyl
cyanoacrylate)-co-(2-octyl cyanoacrylate)] and poly(2-octyl
cyanoacrylate) might be adequate for therapeutical
administration.
Inventors: |
Lee; Yu-Der; (Hsinchu City,
TW) ; Huang; Chi-Yu; (Pingtung County, TW) |
Correspondence
Address: |
Joe McKinney Muncy
PO Box 1364
Fairfax
VA
22038-1364
US
|
Assignee: |
TONG SHEN ENTERPRISE CO.,
LTD.
|
Family ID: |
39498351 |
Appl. No.: |
11/634908 |
Filed: |
December 7, 2006 |
Current U.S.
Class: |
424/489 ;
514/785; 977/788 |
Current CPC
Class: |
A61K 9/5138
20130101 |
Class at
Publication: |
424/489 ;
514/785; 977/788 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 47/14 20060101 A61K047/14 |
Claims
1. A copolymer composition for carrying biological or threapeutical
agents comprising two alkyl cyanoacrylate monomers with the formula
as below: ##STR00004## wherein R=a linear or branched alkyl group
(C.sub.2-C.sub.10).
2. The copolymer composition of claim 1, wherein the content of
each alkyl cyanoacrylate monomer in nanoparticles is more than 0%
(w/w) and less than 100% (w/w).
3. The copolymer composition of claim 1, wherein the nanoparticle
sizes are less than 100 nm with narrow size distribution.
4. The copolymer compositions of claim 1, wherein the biological or
therapeutical carriers control the release rates of encapsulated
drugs.
5. A homopolymer composition for carrying biological or
threapeutical agents comprising 2-octyl cyanoacrylate monomer.
6. The homopolymer compositions of claim 5, wherein the
nanoparticle sizes are less than 100 nm with narrow size
distribution.
7. The homopolymer compositions of claim 5, wherein the biological
or therapeutical carriers have low cytotoxicity and sustainedly
release encapsulated drugs.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions of delivery
nanoparticles. More particularly, the invention relates to
copolymers synthesized by copolymerization of two alkyl
cyanoacrylate monomers.
BACKGROUND OF THE INVENTION
[0002] Biodegradable polymeric nanoparticles had been studied and
applied as efficient carriers for various drugs, peptides and gene
delivery. Many therapeutical agents, particularly those used for
oral or intravenous administrations, might undergo degradation
gradually and be of low medical efficiency. The appropriate
nanoparticles carriers were designed for high loading efficiency
and low cytotoxicity. While encapsulated into the nanoparticles,
these ingredients were fully protected with the biological
stability increased obviously. Recently, various biodegradable
polymers, such as poly(lactic acid) (PLA), poly(glycolic acid)
(PGA), poly(lactid-co-glycolide) (PLGA) and
poly(.quadrature.-caprolactone) (PCL), had been studied extensively
and successfully developed for clinical treatment.
[0003] Poly(alkyl-cyanoacrylate) (PACA) was an well-studied
therapeutically polymeric compositions for surgical as well as
medical applications. PACA was used as alternates or adjuncts to
surgical sutures, meshes and staples or other medical devices in
wound closure. In addition, PACA was described with capabilities of
absorbability and encapsulation for plurality of active agents
(U.S. Pat. No. 6,881,421). A wide range of drugs, such as insulin,
pilcarpine, vaccines, oligonucleotide and anti-tumor drugs, had
been documented to be packaged into the nanoparticles carriers
composed of PACA.
[0004] Based on clinical studies, only treating with high
concentration of anti-cancer drugs might kill cancer cells.
However, these drugs possessed serious cytotoxicity. Treating
anti-cancer drugs with High concentration not only eliminate cancer
but normal cells. Therefore, biodegradable polymeric nanoparticles
were used to protect patients. Presently, PACA nanoparticles,
especially composed of poly(n-butyl cyanoacrylate) (PBCA), was
extensively applied for anti-cancer therapy through intravenous
administration. Depend on particle size and surface characteristics
of PBCA nanoparticles, encapsulants might deliver to different
target organs.
[0005] Conventional techniques of nanoparticles synthesis were by
emulsion homo-polymerizations. The nanoparticles consisted of PACA
were prepared as drug carriers by anion initiated polymerization in
stabilizer-containing acidic solution. The significant advantages
of the prepared nanoparticles included easy preparation,
non-solvent residues remaining and high stability in aqueous
medium.
[0006] In drug delivery system, release rate of therapeutical drug
was controlled by degradation process of nanoparticle carrier. The
degradation process of PACA was that alkanol group was released
during hydrolysis of the ester chain of alkyl cyanoacrylate
monomer. It was indicated in previous studies that several factors,
including particle size, the ester chain length, molecular weight
of polymer, pH of medium and enzyme used, could influence the
degradation rate. The degradation rate of PACA nanoparticles,
compared with other biodegradable polymers such as PLA, PLGA, etc.,
was fast so PACA had less utility as sustained drug release
composition. The PLGA copolymer was able to modulate hydrophobicity
of nanoparticles by adjusting compositions, but PACA homopolymer
was not. Furthermore, the drugs encapsulating and loading
efficiency of nanoparticles was related to the compatibility
between drugs and nanoparticles. Thus, the therapeutical
application of PACA homopolymer might be limited.
SUMMARY OF THE INVENTION
[0007] The present invention provides nanoparticles with better
utility for delivering, therapeutical drugs. These nanoparticles
are of various properties, such as particle size, hydrophobicity,
therapeutical efficiency, cytotoxicity, etc. Because the products
of homopolymerization of alkyl cyanoacrylate have narrow
application for drug delivery or other application, copolymerizaion
of two alkyl-cyanoacrylates was performed. The copolymers composed
of the biodegradable nanoparticles were made from two alkyl
cyanoacrylate monomers. The PACA copolymers can modulate particle
size, hydrophobicity, degradation rate or other characteristics and
be provided for various drugs by adjusting content rations.
Particularly, PACA copolymers are able to regulate hydrolysis rate
to obtain the capability of sustained drug release.
[0008] The nanoparticles composition of the present invention is
related to copolymers polymerized by alkyl cyanoacrylate monomers
with general formula as below:
##STR00001##
[0009] wherein
[0010] R=a linear or branched alkyl group (C.sub.2-C.sub.10).
[0011] The nanoparticles synthesized by anion emulsion
copolymerization are formed in spherical shape with small size,
less than 100 nm in a narrow distribution. Each composition
possesses individual physical and chemical characteristics. Thus,
by modulating the contents of compositions, drugs of wide range can
be delivered in therapeutical administration. In addition, the POCA
nanoparticles, which possess low degradation rate and low
cytotoxicity, are used as more feasible carriers for timed-release
drug.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows a Scanning Electro Micrograph (SEM) picture of
POCA nanoparticles.
[0013] FIG. 2 shows degradation of poly(BCA-co-OCA) nanoparticles
in phosphate buffer solution (PBS; pH7.4) at 37.degree. C.: (a)
butanol and 2-octanol yield (mean.+-.S.D., n=3) presented as a
molar percent of the total BCA and OCA units initially present in
the nanoparticles; (b) butanol concentration (mean.+-.S.D., n=3);
(c) 2-octanol concentration (mean.+-.S.D., n=3).
[0014] FIG. 3 shows in vitro cytotoxicity (mean.+-.S.D., n=3) for
poly(BCA-co-OCA) nanoparticles with varying BCA/OCA (w/w)
composition at the concentration of 10 .quadrature.g/ml.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] Copolymer compositions of the present invention produced
therefrom are used for delivering therapeutical drugs. More
particularly, these nanoparticles can control releasing rate of the
encapsulated active agents by their own appropriate properties
through adjusting their content ratio of copolymerization.
[0016] The nanoparticle copolymers of the present invention are
comprised two alkyl cyanoacrylate monomers with general formula (I)
as below:
##STR00002##
wherein
[0017] R=a linear or branched alkyl group (C.sub.2-C.sub.10).
[0018] The following examples of the two monomers for the synthesis
of cyanoacrylate polymers are illustrative, but not limiting the
scope of the present invention. Reasonable variations, such as
those occur to reasonable artisan, can be made herein without
departing from the scope of the present invention.
[0019] Two monomers produced cyanoacrylate copolymers of general
formula (II) and (III) as below:
##STR00003##
[0020] The copolymers polymerized by BCA and OCA are poly[(n-butyl
cyanoacrylate)-co-(2-octyl cyanoacrylate)] (poly(BCA-co-OCA)). To
prepare the poly(BCA-co-OCA) nanoparticles, monomers were mixed
well and dispersed into polymerization media respectively. Anion
emulsion polymerization was used to prepare the copolymers, and the
detailed methods were described as below:
[0021] The nanoparticles of PBCA, POCA and poly(BCA-co-OCA) with
compositions of BCA/OCA ratio: 100/0, 75/25, 50/50, 25/75 and
0/100% (wt. %/wt. %) were prepared individually by emulsion
polymerization technique.
[0022] About 0.5 g of monomer or monomers mixture of n-butyl
cyanoacrylate and 2-octyl cyanoacrylate was added drop by drop to a
50 ml aqueous solution of 0.01 N hydrochloric acid containing 0.3%
(w/v) of Pluronic F 127 [poly(ethylene oxide)-poly(propylene
oxide)-poly(ethylene oxide) triblock copolymer; MW 12,6000 Da]under
stirred with a magnetic stirrer. The mixtures were stirred for 18
hr in a 100 ml double-walled round-bottomed reactor surrounded by a
thermostatic water bath set at 25.degree. C. Then a solution of IN
sodium hydroxide solution was added to neutralize the suspension,
followed stirring for another 30 min. The nanoparticles formed were
separated by ultracentrifugation at 60,000.times.g for 60 min (CP
100 MX, Hitachi, Japan) and redispersed in water and
lyophilized.
[0023] Freeze-dried nanoparticles were dissolved in acetone (20
mg/ml). Then water was added in steadily until the color of the
solution changed to milky white. The copolymer was collected by
centrifugation at 10,000.times.g for 10 min. the purified copolymer
was obtained by repeating the procedure three times and
collected.
[0024] The collected polymerization products were observed by
scanning electron microscopy (SEM) analysis. SEM was conducted
using a FESEM; Hitachi S-4700. Samples of collected products were
placed on a 400 mesh carbon coated with cooper grid. After drying,
the samples were observed at 15 kV. The prepared nanoparticles were
spherical shape with narrow distribution and did not show any
aggregation (FIG. 1).
[0025] Then, these products were processed several characteristics,
including particle size, polydispersity index (PI) and zeta
potential.
[0026] The particle size and the zeta potential of nanoparticles
were measured by photon correlation spectroscopy (PCS; Zetasizer
3000, Malven Instruments, Malvern, UK) at 25.degree. C. The
scattered light of wavelength 633 nm was detected at an angle of
90.degree.. The nanoparticles dispersion was diluted by water to an
adequate concentration to facilitate measurement. The mean size of
hydrodynamic particle was represented as the value of z-average
size. The width of the size distribution was indicated by
polydispersity index. The results are presented in table 1
below.
TABLE-US-00001 TABLE 1 Feed composition (wt. %) Particle
Polydispersity Zeta potential BCA OCA size (nm) index (mV) 100 0
74.3 .+-. 1.0 0.121 -21.6 .+-. 0.6 75 25 84.3 .+-. 3.1 0.104 -25.0
.+-. 0.1 50 50 89.6 .+-. 3.4 0.103 -24.9 .+-. 2.1 25 75 94.5 .+-.
0.6 0.082 -25.2 .+-. 1.3 0 100 98.1 .+-. 0.3 0.076 -27.7 .+-.
0.5
[0027] Particle size, polydispersity index and zeta potential were
influenced by the ration of BCA/OCA.
[0028] While OCA weight content increased, the particle size of
nanoparticles increased cooresponding, but the values of
polydispersity index decreased. The OCA molecules containing octyl
groups were more hydrophobic and nonpolar than BCA molecules. High
polarity of the polymer tended to stabilize the surface energy of
particles. Therefore, the nanoparticles size increased with
increasing the content of OCA. In previous studies, the degree of
second nucleation was low for hydrophobic monomer in emulsion
polymerization. So the presence of OCA decreased the polydispersity
index of nanoparticles. In addition, zeta potential is the
electrostatic potential of particle surface generated by ions
accumulation. The negative charge increased slightly as the content
of OCA increased.
[0029] The degradation rate of nanoparticles was analyzed by the
concentration of alkanol hydrolyzed from poly(alkyl
cyanoacrylate).
[0030] Lyophilized nanoparticles were redispersed in pH 7.4 PBS at
a concentration of 2 mg/ml and placed in a shaking incubator (60
r.p.m.) at 37.degree. C. At different time intervals, the
nanoparticles were separated from dispersion media by
ultracentrifugation (CP100 MX, Hitachi, Japan) at 60,000.times.g
for 60 min. A 5 ml sample of supernatant was withdrawn from the
dispersion, and mixed with 1.quadrature.1 n-hexanol (internal
standard). This solution was extracted by 2.5 ml of diethyl ether
which was then injected into gas chromatography (HP5890, USA)
coupled with a mass spectrometer (HP 5972, USA). The GC column used
was a DB-5MS (39 m.times.0.25 mm i.d. and a film thickness 0.25
.quadrature.m) capillary column. Carrier gas was helium at a flow
rate of 1.5 ml/min. The oven temperature was ramped from 40 to
300.degree. C. and held for 3 min. The temperature of the injector
and detector were set as 220 and 280.degree. C., respectively.
[0031] The concentrations of alkanols were determined from the
calibration curves. Standard solutions of 0.003, 0.01, 0.05, 0.1
and 0.2 mg/ml of alkanols were prepared separately for calibration.
The peak area ratios (alkanol/n-hexanol) were analyzed from
chromatographic patterns against the concentration of the
respective calibration standards.
[0032] The degradation rates of poly(BCA-co-OCA) nanoparticles were
presented in FIG. 2. The value of degradation rate of POCA was much
lower than PBCA only (FIG. 2a). As OCA content raised, in addition,
the concentrations of the yield alknols significantly decreased
(FIG. 2b and FIG. 2c). Therefore, the hydrolytic rate of
poly(BCA-co-OCA) nanoparticles could be modulated to be of wide
range by adjusting the content of OCA in the copolymer. The
presence of OCA only, particularly, possesses the lowest alkanols
concentration and degradation rate. While active agents
encapsulated into OCA nanoparticles, these encapsulants might be
release sustainedly.
[0033] To evaluate the in vitro cytotoxicity of the
poly(BCA-co-OCA) nanoparticles was used the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay for viability of the human foreskinon fibroblastic HS 68
cells viability for 3 days. Sterile nanoparticles suspension were
diluted by Dulbecco's modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin
(10.sup.4 IU/ml) to the concentration of 10 .quadrature.g/ml.
Fibroblastic cells were seeded at a density of 5.times.10.sup.4
cells per well in a 24-well plate, and grew for 24 hr. Thereafter,
the cells were washed with PBS (pH 7.4) and incubated with diluted
nanoparticles suspension for 3 days at 37.degree. C. and 5% (v/v)
CO.sub.2. After incubation, the upper medium was carefully removed
and cells were washed twice with PBS. Then, 1 ml MTT solution (0.5
mg/ml) was added to each well and incubated for another 4 hr. The
intracellular blue formazon salt metabolizing the MTT by live cells
was dissolved by adding 1 ml dimethylsufoxide. The absorbance
values were measured by a multiwell microplate reader (SUNRISE TS,
TECAN) at a wavelength of 570 nm. The relative cell viability (%)
in comparison with control well containing cell culture medium
without nanopartiles was calculated by the following equation:
relative cell viability ( % ) = [ absorbance ] test [ absorbance ]
control .times. 100 ##EQU00001##
[0034] The results of the in vitro cytotoxicities of PBCA, POCA and
poly(BCA-co-OCA) nanoparticles were presented in FIG. 3.
[0035] Comparing with the PBCA, POCA nanoparticles did not affect
the viability of human foreskin fibroblasts, indicating their low
cytotoxicity. However, except POCA nanopartilces only, there was
little difference between the toxicities of the copolymer
nanoparticles. These results indicated that the cytotoxicity of
POCA nanoparticles was quite low compared to PBCA
nanoparticles.
[0036] In one embodiment of the present invention, POCA
nanoparticles might be more feasible to apply in therapeutical
administration. Comparing with other copolymers, POCA nanoparticles
obtained the lowest degradation rate and rare cytotoxicity during
hydrolysis. Concerning with medical safety, therefore, POCA was
better used in production of therapeutically delivery
nanoparticles.
[0037] Furthermore, the contents of monomers polymerized into
poly(BCA-co-OCA) were not limited only BCA/OCA ratio: 75/25, 50/50
and 25/75% (w/w), but including 0/100 above to 100/0 below
%(w/w).
[0038] While the invention has been described by way of examples
and in terms of the preferred embodiments, it is to be understood
that the invention is not limited to the disclosed embodiments. On
the contrary, it is intended to cover various modifications as
would be apparent to those skilled in the art. Therefore, the scope
of the appended claims should be accorded the broadest
interpretation so as to encompass all such modifications.
* * * * *